Genetic manipulation of Plants

The bacterium that causes crown gall disease in plants has a natural vector for transformation of desirable traits from one plant to another.

Plant Gene Transfer via Agrobacterium

T-DNA

There are Two Major Methods of Plant Gene Transfer

Agrobacterium

A unique bacterial species

Plant-Fungal-Animal Transformation

Agrobacterium tumefaciens1. Soil bacterium closely related to Rhizobium.2. Causes crown gall disease in plants (dicots).

3. Infects at root crown or just below the soil line.

4. Can survive independent of plant host in the soil.

5. Infects plants through breaks or wounds.

6. Common disease of woody shrubs, herbaceous plants, particularly problematic with many members of the rose family.

7. Galls are spherical wart-like structures similar to tumors.

Opine Biosynthesis

1. Within tumor tissues, the synthesis of various unusual amino acid-like compounds are directed by genes encoded on the integrated plasmid.2. The type of opine produced is specified by the bacterial T-DNA

3. Opines are used by the bacteria as a carbon (nutrient) source for growth.

4. Opine catabolism within bacteria is mediated by genes encoded on the Ti plasmid.

Only known natural example of DNA transport between Kingdoms

1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid

2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA).

T-DNA

The T-DNA is transferred from the Bacteria into the Nucleus of the Plant

1. Stably integrates (randomly) into the plant genome.

2. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell.

3. Synthesis of plant growth hormones (auxins and cytokinins) neoplastic growth (tumor formation)

Agrobacterium tumefaciens

plasmid DNA

Plasmid DNA is cut open with an enzyme.

chromosomalDNA

A specific gene is “cut out” of thedonor DNA using the same enzyme.

New gene isinserted intothe plasmid.

Plasmid is transformedinto Agrobacterium.

When mixed with plantcells, Agrobacteriumduplicates the plasmid.

The new gene is transferredinto the chromosomal DNAof the plant cell.

When the plant celldivides, each daughtercell receives the newgene, giving the wholeplant a new trait.

Overview of the Infection Process

Crown gall tumors

a natural a natural example of example of genetic genetic engineering.engineering.

Agrobacterium/plant interactions

opines

Agrobacterium at wound site

transfers T-DNA to plant cell.

Agrobacterium in soil use opines as nutrients.

Genes required to breakdown Genes required to breakdown opinesopines for use as a for use as a nutrient source nutrient source are harbored on the are harbored on the Ti plasmidTi plasmid in addition to in addition to virvir genes essential for the genes essential for the excisionexcision and and transporttransport of the of the T-DNA T-DNA to the to the wounded plant cellwounded plant cell..

T-DNA

vir genes

opine catabolism

pTipTi~200 kb~200 kb

tra

for transfer to the plant

bacterial conjugation

23 kb23 kb

1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas).

2. Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue.

Ti plasmids can be classified according Ti plasmids can be classified according to the opines producedto the opines produced

3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out.

CNH(CH2)2CHCO2H

NH

HO2C(CH2)2CHCO2H

H2N

HN

(Nopaline)

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

Ti plasmids and the bacterial Ti plasmids and the bacterial chromosome act in concert to transform chromosome act in concert to transform

the plantthe plant

3. T-DNA encodes genes for opine synthesis and for tumor production.

4. occ (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

vir genes

opine catabolism

pTipTitra

for transfer to the plant

bacterial conjugation

Agrobacterium chromosomal DNAAgrobacterium chromosomal DNA

chvA chvB

pscA

oriV

T-DNA-inserts into plant genome

Generation of the T-strandGeneration of the T-strand

overdrive

Right Border

Left Border T-DNA

virD/virC

VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

5’

Generation of the T-strandGeneration of the T-strand

Right border

Left border

DvirD/virC

gap filled inT-strand

T-DNA

virE

1. Helicases unwind the T-strand which is then coated by the virE protein.

2. ~one T-strand produced per cell.

1. Transfer to plant cell.2. Second strand synthesis3. Integration into plant chromosome

Right border

Left border

DT-strand coated with virE

T-DNA

virD nicks at Left Border sequence

The The virvir region is responsible for the transfer region is responsible for the transfer of T-DNA to the wounded plant cell.of T-DNA to the wounded plant cell.

receptor for acetyl-syringone

positive regulator for other vir genes

virA

constitutive

virG

virA is the sensor.

membraneactivated virG

Note: activated virG causes its own promoter to have a new start point with increased activity.

virA is the sensor.

bacterialmembrane

Acetylsyringone is produced by wounded plant cells (phenolic compound).

triggers auto-phosphorylation of virA

1 2

P3

virG

virA

virG activates transcription

from other vir promoters.

VirA phosphorylates virG which causes virG to become activated. virG is the effector.

Asg

AsgP

The The virvir region is responsible for the transfer region is responsible for the transfer of T-DNA to the wounded plant cell.of T-DNA to the wounded plant cell.

ssDNA binding protein. Binds T-strand.

virA virGvirB virCvirD virE

sensor effector

endo-nuclease nicks T-DNA

Binds overdrive DNA.

membrane protein; ATP-binding

Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria.

Generation of the T-strandGeneration of the T-strand

overdrive

Right Border

Left Border T-DNA

virD/virC

VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

5’

Generation of the T-strandGeneration of the T-strand

Right border

Left border

DvirD/virC

gap filled inT-strand

T-DNA

virE

1. Helicases unwind the T-strand which is then coated by the virE protein.

2. ~one T-strand produced per cell.

1. Transfer to plant cell.2. Second strand synthesis3. Integration into plant chromosome

Right border

Left border

DT-strand coated with virE

T-DNA

virD nicks at Left Border sequence

1. VirB1 may have local lytic activity that allows assembly of the transporter at specific sites in the cell envelope.

2. The processed VirB1* peptide is secreted through the outer membrane by an unknown mechanism.

3. The structural components of the pilus are VirB2 and VirB5.

4. Complexes of VirB7/9, formed by disulfide bridges, may initiate assembly of the VirB channel.

5. The exact role of VirB3, 4, 6, 8, 10 and 11, and VirD4 in the transporter apparatus is unknown.

Assembly of the Agrobacterium T-Complex Transport Apparatus

6. VirD4, VirB4 and VirB11 have nucleotide-binding motifs that are essential for their activity.

7. The T-complex, consisting of a ss copy of T-DNA bound to VirD2 and coated with VirE2, is exported through the transport apparatus.

SP, signal peptide; SPI, signal peptidase I.

(a) The pilus has not contacted the surface of the recipient plant cell and the apparatus is unable to transport T-complex.

(b) The pilus has contacted a receptor (?) on the surface of the recipient plant cell. This induces the VirB transporter, perhaps via a change in conformation, so that it is now competent to transfer the T-complex to the plant cell cytoplasm.

OM, outer membrane; IM, inner membrane; CW, plant cell wall; PM, plasma membrane.

Model for contact-dependent activation of the T-complex transport apparatus

Agrobacterium can be used to transfer DNA into plants

pTi-based vectors for plant pTi-based vectors for plant transformation:transformation:

2. Early shuttle vectors integrated into the T-DNA; still produced tumors.

1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium.

E. coli Agrobacterium

pTiShuttle plasmid

conjugation

Several hundred tumors containing foreign gene can be grown for experimental purposes.

Transformed sunflower seedlings

Harvest time!

3 weeks after inoculation

Transformation of Arabidopsis plants

Dip floral buds in 1 ml of Agrobacterium culture for 5 to 15 min.

Detergent added to allow bacteria to infiltrate the floral meristem.

Transformation of Arabidopsis plants

700 to 900 seeds per plant.

Germinate on kanamycin plates to select transformants.

10 to 20 transformed plants per plant.

10 day old seedlings

MiniTi T-DNA based vector for plants

1. 1. Binary vectorBinary vector: the : the virvir genes genes required for mobilization and required for mobilization and transfer to the plant reside transfer to the plant reside on a on a modified pTimodified pTi..

2. consists of the 2. consists of the right and left right and left border sequencesborder sequences, a , a selectable marker selectable marker (kanomycin (kanomycin resistance) and a resistance) and a polylinkerpolylinker for insertion of a foreign for insertion of a foreign gene.gene.

Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene.

miniTi

MiniTi T-DNA based vector for plants

modified Ti plasmid

a binary vector system

oriVoriV

virvir

T-DNA deleted

2

LBRB

oriori

kanr polylinker

miniTiminiTi

bombom1

bom = basis of mobilization

Transfer of miniTi from E. coli to Agrobacterium tumefaciens

Triparental mating:Triparental mating:

bombom site for site for mobilizationmobilization

miniTi;miniTi;kan resistancekan resistance

E. coliAgrobacteriumstr resistant

pRK2013;pRK2013;kan resistancekan resistance

contains tratra genes

modified pTimodified pTi

15A ori;15A ori;E. coli or Agrobact.E. coli or Agrobact.

ColE1 oriColE1 ori

tra bom

Ti oriVTi oriV

Steps in the mating 1-2:

Triparental mating:Triparental mating:

pRK2013;pRK2013;kan resistancekan resistance

contains tratra genes

tra

ColE1 oriColE1 ori

bomtra1

2

E. coli

Helper plasmid (pRK2013) mobilizes itself into 2nd E. coli strain containing miniTi.

miniTi;miniTi;kan resistancekan resistance

Steps in the mating 2-3:E. coli

miniTi;miniTi;kan resistancekan resistance

Agrobacterium

Helper plasmid mobilizes itself and the miniTi into Agrobacterium.

2 miniTi 3

pTipRK2013

miniTi

pRK2013 can not replicate.

pRK2013

Selection of Agrobacterium containing the miniTi on strep/kan plates

miniTi;miniTi;kankan resistance resistance

pRK2013;pRK2013;kankan resistance resistance modified pTimodified pTi

Agrobacteriumstr resistantAgrobacteriumstr resistantplate on str and kan media

tra

str rbom

can not replicate

pTiminiTi

pRK2013 kanrstr r

Alternate Methods of Transforming Plants: Particle Bombardment

One way of physically introducing DNA into cells is with a particlegun.              

•Very tiny DNA-coated metal particles are suspended in a drop on a macroprojectile.

•A discharge (from a gunpowder explosion or from breakage of a membrane enclosing a pressurized chamber) impels the macroprojectile.

•The macroprojectile is stopped by a stopping plate, but the microprojectiles continue into the tissue below.

•The DNA introduced with the particles is expressed

1. DNA- or RNA-coated gold/tungsten particles are loaded into the gun and you pull the trigger.

Particle Bombardment using the Gene Gun

2. A low pressure helium pulse delivers the coated gold/tungsten particles into virtually any target cell or tissue.

3. The particles carry the DNA cells do not have to be removed from tissue in order to transform the cells

4. As the cells repair their injuries, they integrate their DNA into their genome, thus allowing for the host cell to transcribe and translate the transgene.

Agrobacteria are biological vectors for introduction of genes into plants.

•Agrobacteria attach to plant cell surfaces at wound sites.

•The plant releases wound signal compounds, such as acetosyringone.

•The signal binds to virA on the Agrobacterium membrane.

•VirA with signal bound activates virG.

Summary

•Activated virG turns on other vir genes, including vir D and E.

•vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left border. The left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell

•Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border.

•The T-DNA is transferred to the plant cell, where it integrates in nuclear DNA.

•T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.